Difference between revisions of "Arsenic Prototype 4.0"
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===Making the Outer Housing=== | ===Making the Outer Housing=== | ||
Revision as of 11:59, 5 February 2016
The Arsenic prototype v4.0 is the Winter School 2016 kit version of prototype 3.0.
Contents
- 1 Principles
- 2 Tools
- 3 Safety
- 4 Materials
- 5 Before Starting
- 6 Step by Step Buildling
- 7 Issues and Observations
- 8 Links other References
Principles
The basis of the prototype remains the same as the prototype 3.0- the bioreporter, a GMO bacteria expressing eGFP (Green Fluorescent Protein) is incubated with a water sample, where the fluorescence is detected optically and can be quantified in order to measure the concentration of Arsenic in the water
- A vial containing the water sample we want to test is positioned on a socket through which a fluorescent excitation LED (blue 488nm for eGFP) passes.
- GFP absorbs this blue light (λ=475 nm) and emits green light (λ=504 nm) which is detected by a photosensor on which the light is concentrated with the help of two lenses that avoid loss of intensity.
- A long-pass filter allows only the eGFP fluorescence signal to reach the photosensor.
- The intensity of the fluorescence is a function of the number of bacterias in the sample. This can be quantified undirectly by measuring the Turbidity of the sample.
- A red LED is placed in-line with the photosensor to measure the transmittance, which can be converted into turbidity. The measurement of turbidity will allow us to normalize our results with respect the density of bacterias.
Such a system is easily implemented using a microcontroller, a minimalist computer. A microcontroller has a number of GPIO (general purpose input output). When turned as an output, a GPIO is a terminal that can be programmatically set to a voltage of 5V (high state, logical 1), or 0V (low state, logical 0). This can be used to turn on or off external components such as LEDs. When used as an input, we can read the light sensor from them for example. The microcontroller is programmed in C code. It incorporates also timers that allow to keep track of time.
We use an Arduino board to control this device. The microcontroller on the Arduino board is programmed to carry out the following operations.
- Wait for button to be pushed.
- Turn on the blue LED for a predetermined amount of time.
- Readout the value of the light sensor.
- Turn off the blue LED.
- Turn on the red LED for a predetermined amount of time.
- Readout the value of the light sensor.
- Display the two values on the screen of the device.
The main electronic components of the device are as follows.
- Arduino board: the brain of the device.
- Transistors: the GPIO cannot provide enough current to drive LEDs directly. We use instead transistors. Transistors are like electrically controllable switches. It is a 3-terminal device. When provided with 0V or 5V on one terminal, the two remaining terminals are connected or disconnected, respectively (or vice versa depending on transistor type). The transistor size is proportional to how much current it must let through. We need a large one for the blue LED as it is very bright and has a high current.
- LED at 600 nm: to measure optical density.
- LED at 485 nm: to excite eGFP.
- A light sensor: the sensor we use is a light-to-frequency sensor. It means that it outputs a square signal with a frequency proportional to the light intensity on the photosensitive part of the sensor.
- A screen: to display results.
- Buttons and potentiometer: for the user to interact with the device.
With the measures taken by the device, compared against a standard curve of water containing known arsenic concentrations, one can determine the concentration of arsenic in the sample and so know if the water is drinkable or not.
In summary, the most current version consists of:
lasercut chassis blue LED for eGFP fluorescence excitation at 90 degrees from detector red LED for transmittance measurements in line with detector vial holder matching the vial approved by the Swiss authorities light to frequency detector a filter to block excitation light LCD screen read out based on the arduino
Tools
- Soldering iron
- Tin
- Knippers
- Plier
- Screwdriver phillips #1
- Vice (for crimping the ribbon cable)
Safety
- Don't burn the wires on the table with the soldering iron
- Mind your fingers
- When knipping the legs of the components, hold the loose end so that it doesn't fly into someone's eye.
- Apply common sense
Materials
For the full BOM including reference numbers see this sheet.
LCD Shield
- 1x LCD 8x2 HDM08216L-3-L30S
- 1x resistor 1K (R1)
- 1x Trimpot 10K
- 2x Tactile switch 17mm (S1, S2)
- 1x Potentiometer
- 1x Micromatch connector 10 pin SMD female 8-338069-0
- 1x 1x40 pin header straight long (17mm)
- 1x 2x8 pin header straight
LED Driver board
- 1x LED 485nm (blue) SMD XPEBBL-L1-0000-00Z01
- 1x P-channel MOSFET TO-220
- 1x P-channel MOSFET TO-92
- 1x Micromatch connector 10 pin SMD female 8-338069-0
- 1x Resistor 5 ohm 3W
- 1x Resistor 150 Ohms 1/4W
- 1x 1x3 pin header 90 degree bent
- 1x 1x2 pin header 90 degree bent
Optical density (red LED)
- 1x red LED 5mm
- 1x 1x2 pin header straight
Light sensor
- 1x Light To Frequency Converter – TSL235R
- 1x 1x3 pin header straight
Optics
- 2x [Plastic Biconvex Lenses LPP1009
- 1x 1.5 x 1cm 510nm long pass acrylic filter 510FAP5050
Miscellanea
- 1x Arduino UNO Rev 3
- 1x 2.1mm DC barrel jack
- 1x 9V battery snap and contact connector
- 5x Jumper cables female-to-female 10 cm
- 1x 9V Battery
- 20cm Ribbon cables 1.27 10 wires
- 2x Micromatch connector 10 pin male 8-215083-0
Mechanical
- 12x M3 screws 10mm
- 12x M3 nuts
- 10x M3 washers
Before Starting
Soldering is easy! Learn it!
Step by Step Buildling
Laser Cutting the Inner Scaffold Pieces
at chez hackuarium neighboring @make space of univercité - laser cutter model: Keyland KQG-1060 120W CO2 laser cutter
| mode | speed | power | scan mode | interval | |
| Groove LED (fluo) | scan | 50 (100) | 35 | x_unilat | 0.1 |
| 3mm board | cut | 20 | 100 | ||
| Text and Logo | scan | 250 (100) | 25 | x_unilat | 0.3 |
| Groove Lens | scan | 50 | 12 | x_unilat | 0.1 |
| 2mm board | cut | 35 | 100 |
With these parameters, the 3mm thickness pieces take 5 min to cut, and the 2mm thickness pieces take min.
Results
high-density fiberboard (HDF)
medium-density fiberboard (MDF)
Assembling the Electronics
There are four PCBs for:
- Blue LED (for the eGFP excitation)
- LCD screen
- Red LED (for the transmittance)
- Light to frequency meter (to detect the light)
TIP: if this is your first time soldering, we suggest to start with the LCD screen
Each of the PCBs except the LCD screen PCB are fixed onto the lasercut fiber boards using M3 screws.
Soldering and Mounting
Blue LED
Soldering the pieces:
1. the super bright blue surface mount LED (SMD) piece is manually soldered onto the "backside" of the PCB
2. on the "front side" of the PCB, first solder the resistor
3. clip the legs of the resistor from the back
4. solder the other components (the connectors for the Red LED and the light to frequency meter)
5. Solder in the large MOSFET transistor. Try to bend it so that it stays flat on the PCB, hole aligned.
6. the other MOSFET is soldered into place - the flat side facing the resistor.
7. pay attention to the orientation of the ribbon cable connector (the connector is red in this picture, but black in subsequent, it is the same connector)
8. to place the ribbon cable connector, first apply solder on one pad of the PCB (e.g. the circled pad in previous picture)
9. solder the first foot of the ribbon cable connector, ater soldering remove the sticker atop the connector, then proceed with the rest
10. all except the resistor for the super bright LED is soldered
10. ready for mounting
Now mounting onto the particle board:
1. M3 screws are placed
2. Two washers are placed onto the screws before the PCB
3. the SMD Blue LED should face the particle board, and the PCB is bolted in
LCD screen
This LCD screen / arduino shield will connect to the blue LED PCB with a ribbon cable
Soldering the pieces:
1. take out the Arduino board from its package. Cut a piece of electrical tape and cover the USB connector as shown on the picture.
2. take out the long pin header, break it down into 2 pieces of 6 pins, and 2 pieces of 8 pins. Place all the pins in the female connectors of the Arduino board as shown. The 6-pins go on the side with the analog inputs (A0, A1, etc), and the 8-pins on the digital IO side. You should now be able to place the PCB on top with all pins sticking out
3. place the green PCB board on top of it with the pin headers sticking out at the correct place. Solder all the pin headers in place (in this picture, the resistor is already there, but it is actually easier to do it next)
4. we will now solder the ribbon cable connector just as we did in the previous step (Blue LED steps 8 and 9), again pay attention to orient it as shown here
5. solder resistor 1 on the logo side
6. back to the logo side, place the trimpot on the left, two small buttons S1, S2 on the right, and the large potentiometer on the right. Solder into place from the back
7. solder the pins onto the LCD screen, as the others, solder one, and make sure the placement is good before soldering the other pins
8. LCD screen with view of the pins from the back
9. view of the back with the big button soldered in
9. done soldering this board, onto the next
Red LED
1. first mount the red LED - short leg / flat plastic rim = negative, then solder
2. next, the pins, long pins same side as the LED, then solder
3. ready to mount on the board
4. mounted, view from the PCB side - washers not necessary for the screws
5. mounted, view from the LED side
light to frequency meter
1. place the legs of the light to frequency detector (LFD) in the PCB without bending or soldering, the goal will be to align it with the picture on the PCB
2. use the particle board to align the 'eye' of the LFD, fix already the particle board to the PCB by using two M3 screws and bolts, place a washer between the two boards, verify that the light sensor lies flat on the PCB, if not, push it with a plier or a screwdriver
3. solder the LFD and the long pins same side as the LFD
4. back of the soldered PCB aligned with the particle board
5. place a washer to account of the LFD thickness
6. mounted, view from the PCB side
7. mounted, view from the LFD side
8. take out the yellow filter and prepare to fix it over the LFD - remove the clear film that is protecting the filter plastic
9. use the glue gun to put a dot of glue on both side of the filter, be careful to not put any glue where the other particle boards will come during assembly
10. the filter after being fixed
Battery connector
1. get the DC barrel jack, unscrew the plastic part and drive the wires through it before soldering anything
2. drive the red wire through the central hoop from inside out, and the black wire to the larger outer rim also from inside out, bend back the wires
3. with the help of a 3rd hand, immobilize the connector and solder the wires into place
4. cut one cable from the battery adaptor and solder it on one outer and one middle leg of the switch
5. use a glue gun to stabilize the switch connection
Lens assembly
- Place the two sides next to each other. They should be both marked with the *same* letter (A/A or B/B).
- Place the lens in the groove on one side.
- Place the second side on top, with letters towards the inside for both sides.
- Fix together with 2 screws and 2 bolts.
Vial Stabilizing Ring
1. Either etch a groove on the middle board or place a ring cut from MDF
2. Use wood glue or glue gun to glue onto the middle board - make sure the gluing is clean in the inside so that the vial can sit snugly
Final Assembly
Finally, the Red LED and light to frequency meter will be plugged into the Blue LED board with connectors.
Then the LCD screen is plugged into the Blue LED board with a ribbon cable.
The LCD screen board is ready to be mounted on the arduino.
We are now ready to test it out.
Testing the Electronics
Uploading the arduino code
If you have not done so already, download the arduino open software.
The firmware is in our github repository for the FluoMeter.
Reading the measurements
First test the firmware and the hardware without sample.
- When you push the start button, does the blue LED go on?
- Do you see the measurement on the LCD screen?
- Do you then see the red LED go on?
Then try it with sample with fluorescence
- Do you see changes in the measurement?
Now we are ready for the actual bacterial samples.
Transmittance and absorbance
What we actually measure with the red LED is the transmittance - how much of the red LED light goes through the sample and reaches the other side.
Absorbance (optical density) is then calculated.
where (Red LED captured by the light to frequency meter with sample / with buffers (no bacteria))
Normalizing the Fluorescence Data
As the fluorescence intensity depends also on the number of bacteria present in the vial, it is necessary to normalize for this - especially when comparing between time points, or if toxicity to the bacteria are seen.
Normalized Fluorescence Units = NFU
where B = reading from the blue LED, and A is the absorbance calculated from above.
Making the Outer Housing
What we have built so far leaves the samples exposed to ambient light.
So we need to make outer housing that can mount the LCD screen, house the battery, enclose the scaffold inside, and have easy access to put the samples in and out while keeping the light and optics dark.
Issues and Observations
During the Winter Workshop of February 1-5 2016, here is the list of issues to be addressed on this workshop
- test for "light proofness" for outer box
- more robust casing
- when the battery is first turned on, the first fluorescent reading is much higher than the rest of the readings
- Problem due to interrupt flag not properly cleared. Fix in github.
- battery drains quickly >>> alternatives: use a USB battery charger
- make a USB cable hole in the outer box
- 2ml in the 4ml vial seems to improve eGFP folding, and early detection of the eGFP fluorescence
- improve signal to noise ratio by signal lock-in-amplification
Links other References
